Currently, there is an urgent need to integrate imaging and therapy for the detection and treatment of breast cancer. Of particular concern is the aggressive triple negative subtype which lacks the progesterone and estrogen receptors and epidermal growth factor type 2 receptor (Her-2/neu) and accounts for approximately half of all breast cancer deaths. To this end, we propose developing a molecular image based therapy to assess the localization and extent of breast cancer based on epithelial protein-2 (EMP2) expression. EMP2 is a novel biomarker highly expressed in the majority (63%; n=97) of invasive breast cancer tumors examined compared to healthy mammary epithelium. Its expression is independent of ER, PR, and HER2/neu expression, and high EMP2 expression is observed in over 70% of triple negative breast cancer cases examined. As engineered recombinant antibodies hold great promise for cancer diagnostics and therapy, we have carefully assembled a research team centered at UCLA to develop recombinant EMP2 antibody fragments for both imaging using PET and therapy. We have previously shown that recombinant antibody fragments (diabodies) against EMP2 promote apoptosis both in vitro and in vivo in a number of EMP2 positive tumors, and we have new data that radiolabeled minibodies specificially recognize EMP2 positive tumors using microPET. Moreover, new preliminary data clearly indicate that systemic administration of the EMP2 IgG1 reduces tumor load in human xenograft and orthotopic, syngeneic mouse model systems. In this proposal, with the explicit goal of creating a product to advance for clinical testing, we will determine the optimum full-length antibody and antibody fragment for use in therapy and imaging, respectively. We have already generated a number of these reagents, and our EMP2 antibodies and antibody fragments exhibit human and murine cross-reactivity. In this study, we will define a more complete toxicity profile, including pK, pD, and maximum tolerated dosage of these reagents to validate their usage. As a step towards using engineered EMP2 antibody fragments as an imaging agent, we propose engineering two antibody fragment formats with different pharmacokinetic and clearance characteristics to allow maximization of expression and binding. For therapy, we have created two unique fully human IgG1 antibodies that recognize the second extracellular loop of EMP2. Following validation of the specificity and sensitivity of these fragments in defined human breast cancer model systems, we will integrate therapy of breast cancer using a-EMP2 antibodies or neoadjuvant chemotherapy. Successful completion of the work proposed in this grant submission will identify the optimum strategy for imaging and therapy of breast cancer using EMP2. Given its high expression in a number of gynecological tumors including ovarian and endometrial, these studies will be important to position EMP2 as a viable target for cancers in women. PUBLIC HEALTH RELEVANCE: Despite significant advances in biology and medicine, the incidence and mortality due to breast cancer world- wide is still unacceptably high. Breast cancer remains the 5th most common cause of death in women. Recently, we have identified a protein epithelial membrane protein-2 (EMP2) that is upregulated in 68% of all breast cancer tumors and 73% of triple negative tumors tested. In the studies proposed here, we present a novel and exciting imaging guided therapeutic approach through the targeting of EMP2 in breast cancer.